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High mobility group box 1 protein induction by Mycobacterium bovis BCG.

Hofner P, Seprényi G, Miczák A, Buzás K, Gyulai Z, Medzihradszky KF, Rouhiainen A, Rauvala H, Mándi Y - Mediators Inflamm. (2007)

Bottom Line: The BCG strain resulted in a higher amount of secreted HMGB1 (450 +/- 44 ng/mL) than that of LPS (84 +/- 12 ng/mL) or Staphylococcus aureus (150 +/- 14 ng/mL).BCG and Phorbol -12-myristate -13 acetate (PMA), added together, resulted in the highest HMGB1 secretion (645 +/- 125 ng/mL).Assesment of the pathophysiological role of this late cytokine in mycobacterial infections demands further in vitro and in vivo examinations.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Szeged 6720, Hungary.

ABSTRACT

Unlabelled: High mobility group box 1 protein (HMGB1), a nuclear protein, is a critical cytokine that mediates the response to infection, injury, and inflammation. The aim of our study was to elaborate a reliable in vitro model to investigate whether Mycobacterium bovis BCG is able to induce HMGB1 secretion from the monocytic U-937 cells. Western blot technique was applied for the detection of HMGB1 from supernatants of cells, following induction with Mycobacterium bovis BCG. Densitometric analysis revealed higher concentrations of HMGB1 in cell supernatants stimulated with BCG than in the supernatants of the control, nonstimulated cells. Further quantitation of the secreted HMGB1 was performed by ELISA. The BCG strain resulted in a higher amount of secreted HMGB1 (450 +/- 44 ng/mL) than that of LPS (84 +/- 12 ng/mL) or Staphylococcus aureus (150 +/- 14 ng/mL). BCG and Phorbol -12-myristate -13 acetate (PMA), added together, resulted in the highest HMGB1 secretion (645 +/- 125 ng/mL). The translocation of the HMGB1 towards the cytoplasm following infection of cells with BCG was demonstrated by immunofluorescence examinations.

Conclusion: Our pilot experiments draw attention to the HMGB1 inducing ability of Mycobacterium bovis. Assesment of the pathophysiological role of this late cytokine in mycobacterial infections demands further in vitro and in vivo examinations.

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Related in: MedlinePlus

Mass spectral identification of recombinant human HMGB-1 from E. coli. Database search with MS data identified the 30 kDa band as HMGB-1 protein, which was confirmed by MS/MS spectra of m/z 1128.56 matching the sequence YEKDIAAYR of HMGB-1 and 1768.76matching the sequence GSSHHHHHHSSGLVPR of hexahistidine peptide (His-tag). 79% of the masses detected matched this protein. Sequence positions are in square brackets.
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fig2: Mass spectral identification of recombinant human HMGB-1 from E. coli. Database search with MS data identified the 30 kDa band as HMGB-1 protein, which was confirmed by MS/MS spectra of m/z 1128.56 matching the sequence YEKDIAAYR of HMGB-1 and 1768.76matching the sequence GSSHHHHHHSSGLVPR of hexahistidine peptide (His-tag). 79% of the masses detected matched this protein. Sequence positions are in square brackets.

Mentions: The identity of both proteins was confirmed unambiguously. In-gel tryptic digestionand peptide extraction was followed by mass spectrometry. MALDI-TOF analysis ofthe unfractionated tryptic digest of the appropriate gel band identified 79%of the masses detected as predicted tryptic cleavage products of human HMGB1 (Figure 2). These peptides represented approximately 56% of the protein sequence (see bold letters). The identity of 4 peptides: Lys30-Lys43, His31-Lys43, Ile113-Lys127, and Tyr155-Arg163 was further confirmed by CID analyses. Masses, corresponding to predicted His-tag tryptic peptides, were also detected, their identity was confirmed by PSD analysis. Similarly, 82% of the masses detected in the second positive standard sample (i.e., recAtn) matched predicted tryptic peptides of the rat HMGB1. The identity of m/z 1520.76 as the predicted Ile113-Lys127 peptide was further confirmed by PSD analysis. These peptides represented approximately 47.6% of the protein sequence (Figure 3, see bold letters).


High mobility group box 1 protein induction by Mycobacterium bovis BCG.

Hofner P, Seprényi G, Miczák A, Buzás K, Gyulai Z, Medzihradszky KF, Rouhiainen A, Rauvala H, Mándi Y - Mediators Inflamm. (2007)

Mass spectral identification of recombinant human HMGB-1 from E. coli. Database search with MS data identified the 30 kDa band as HMGB-1 protein, which was confirmed by MS/MS spectra of m/z 1128.56 matching the sequence YEKDIAAYR of HMGB-1 and 1768.76matching the sequence GSSHHHHHHSSGLVPR of hexahistidine peptide (His-tag). 79% of the masses detected matched this protein. Sequence positions are in square brackets.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233895&req=5

fig2: Mass spectral identification of recombinant human HMGB-1 from E. coli. Database search with MS data identified the 30 kDa band as HMGB-1 protein, which was confirmed by MS/MS spectra of m/z 1128.56 matching the sequence YEKDIAAYR of HMGB-1 and 1768.76matching the sequence GSSHHHHHHSSGLVPR of hexahistidine peptide (His-tag). 79% of the masses detected matched this protein. Sequence positions are in square brackets.
Mentions: The identity of both proteins was confirmed unambiguously. In-gel tryptic digestionand peptide extraction was followed by mass spectrometry. MALDI-TOF analysis ofthe unfractionated tryptic digest of the appropriate gel band identified 79%of the masses detected as predicted tryptic cleavage products of human HMGB1 (Figure 2). These peptides represented approximately 56% of the protein sequence (see bold letters). The identity of 4 peptides: Lys30-Lys43, His31-Lys43, Ile113-Lys127, and Tyr155-Arg163 was further confirmed by CID analyses. Masses, corresponding to predicted His-tag tryptic peptides, were also detected, their identity was confirmed by PSD analysis. Similarly, 82% of the masses detected in the second positive standard sample (i.e., recAtn) matched predicted tryptic peptides of the rat HMGB1. The identity of m/z 1520.76 as the predicted Ile113-Lys127 peptide was further confirmed by PSD analysis. These peptides represented approximately 47.6% of the protein sequence (Figure 3, see bold letters).

Bottom Line: The BCG strain resulted in a higher amount of secreted HMGB1 (450 +/- 44 ng/mL) than that of LPS (84 +/- 12 ng/mL) or Staphylococcus aureus (150 +/- 14 ng/mL).BCG and Phorbol -12-myristate -13 acetate (PMA), added together, resulted in the highest HMGB1 secretion (645 +/- 125 ng/mL).Assesment of the pathophysiological role of this late cytokine in mycobacterial infections demands further in vitro and in vivo examinations.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Szeged 6720, Hungary.

ABSTRACT

Unlabelled: High mobility group box 1 protein (HMGB1), a nuclear protein, is a critical cytokine that mediates the response to infection, injury, and inflammation. The aim of our study was to elaborate a reliable in vitro model to investigate whether Mycobacterium bovis BCG is able to induce HMGB1 secretion from the monocytic U-937 cells. Western blot technique was applied for the detection of HMGB1 from supernatants of cells, following induction with Mycobacterium bovis BCG. Densitometric analysis revealed higher concentrations of HMGB1 in cell supernatants stimulated with BCG than in the supernatants of the control, nonstimulated cells. Further quantitation of the secreted HMGB1 was performed by ELISA. The BCG strain resulted in a higher amount of secreted HMGB1 (450 +/- 44 ng/mL) than that of LPS (84 +/- 12 ng/mL) or Staphylococcus aureus (150 +/- 14 ng/mL). BCG and Phorbol -12-myristate -13 acetate (PMA), added together, resulted in the highest HMGB1 secretion (645 +/- 125 ng/mL). The translocation of the HMGB1 towards the cytoplasm following infection of cells with BCG was demonstrated by immunofluorescence examinations.

Conclusion: Our pilot experiments draw attention to the HMGB1 inducing ability of Mycobacterium bovis. Assesment of the pathophysiological role of this late cytokine in mycobacterial infections demands further in vitro and in vivo examinations.

Show MeSH
Related in: MedlinePlus